HPV-associated Disease
The human papillomaviruses (HPVs) are small DNA viruses that infect cells of the cutaneous and mucosal epidermis. Over 80 different HPV genotypes have been characterized. Some types, such as HPV-1, -2, -3, -4 and -10, cause cutaneous lesions known as warts or papillomas. These growths are benign and self-limiting, and are found on the hands and feet of 7-10% of the general population. Of greater medical concern are those HPV types that infect the anogenital tract. These genotypes are designated as either “low-risk” or “high-risk” based on their correlation with malignant progression.
So-called low-risk HPVs are associated with genital warts, or condyloma acuminata. For instance, HPV types 6 and 11 are found in more than 90% of benign genital lesions, and very rarely associated with malignant transformation. However, they nonetheless represent a serious public health problem. Approximately 1% of sexually active adults in the U.S.A. have visible genital warts, but in many more cases the infection is sub-clinical. In fact, an estimated further 15% of people aged 15-49 display molecular evidence of HPV infection, in the form of viral DNA detectable by polymerase chain reaction (PCR) assay. Indeed, HPV is ranked as the most common sexually-transmitted viral agent in the U.S.A. and U.K., and its incidence is increasing steadily.
Infection with high-risk HPV types such as 16, 18, 31 and 33, has been strongly linked to the development of anogenital malignancies, most notably cervical cancer. In fact, HPV types 16 and 18, while rarely found in benign genital lesions, are detectable in about 70% of all invasive carcinomas of the cervix. The link between HPV and anogenital cancer is well documented—recent studies have found that almost 90% of cervical carcinomas contain HPV DNA.
Current Therapies and the Need for a Virus-Specific Treatment
In spite of the pervasiveness of HPV infection and its possibly life-threatening consequences, no virus-specific inhibitor has yet been described. Antiviral drug discovery for HPV has proven quite difficult thus far as a result of difficulties encountered in propagating the virus in the laboratory.
All current therapies for HPV infection rely on the non-specific destruction or removal of infected tissue. Accepted surgical procedures include the use of dry ice, liquid nitrogen, CO2 laser therapy, electrocautery or local excision. Various cytotoxic agents are also used to destroy tissue, such as salicylic acid, tricholoroacetic acid, podophyllin, colchicine, bleomycin and cantharidine.
While the risk of cancer makes these procedures the most prudent for the treatment of high-risk HPVs, less invasive treatments are being sought to manage the low-risk genotypes. Compounds that stimulate the immune system have been investigated with the goal of reproducing the spontaneous regression often seen with benign lesions. Imiquimod, such an immune response modifier, has recently passed clinical trials and been approved for treatment of HPV-associated genital warts.
Patients with genital warts often experience high recurrence rates—usually 30-90%-following non-specific treatments such as surgery. Such poor efficiency is a result of the incomplete elimination of HPV DNA, or the presence of virus in normal-appearing tissue adjacent to the papilloma. Obviously, there is a substantial need for an effective, virus-specific therapy for HPV infection, which has thus far gone unmet.
Viral DNA Replication and E1
Semi-conservative DNA replication is an intricate process mediated by many enzymes and accessory proteins. Helicases are enzymes that function during DNA replication, catalyzing the unwinding of duplex DNA ahead of the replication fork. They are very common in prokaryotic and eukaryotic cells, as well as most viruses. The exact mechanism by which helicases convert the binding and hydrolysis of ATP into mechanical energy to power the unwinding of DNA and their own simultaneous motion along the nucleic acid stand is still not completely understood.
The 72 kDa HPV E1 protein has been classified as a member of helicase superfamily III along with the T antigen of Simian Virus 40 (SV40 TAg), with which it is structurally and functionally homologous. E1 and Tag belong to a noteworthy subgroup of viral DNA helicases which have the ability to recognize and bind specific DNA sequences at the viral origin of replication (ori). Also, while most DNA helicases require a region of single-stranded DNA for entry, these proteins can initiate unwinding from completely double-stranded DNA, provided it contains an ori.
Molecular Events at the HPV Origin of Replication
Human papillomaviruses contain approximately 8 kb of double-stranded circular DNA. In the basal cells of the epidermis, the genome is replicated and maintained extra-chromosomally at a steady-state level of about 20-100 copies per cell. High-level amplification of the genome only occurs once the cell has terminally differentiated and migrated to the upper layers of the epithelium.
In a cell-free DNA replication system, the E1 protein can direct origin-specific DNA replication by itself at sufficient concentrations, when provided with the full complement of host replication proteins including the DNA polymerase α primase enzyme. However, replication is greatly stimulated by the viral E2 protein, and at limiting concentrations of E1 the in vitro replication becomes completely E2-dependent. This is a consequence of E1 having a relatively low affinity for its DNA binding site. E2 helps to localize E1 to the origin by acting as an accessory protein. The E1 and E2 binding sites at the viral ori are in close proximity, falling within about 100 bp of each other. The carboxy terminus of E2 binds its palindromic site on DNA, while the amino terminus binds E1, thus bringing E1 to its binding site.
E1 as a Target for Antiviral Therapy
Recently, pharmaceutical companies have been able to substantially expand and accelerate their antiviral compound screening programs as a consequence of advances in molecular biology. Viruses are now routinely examined at the molecular level to find specific inhibitors of virus-encoded gene products.
For several viruses, enzymes such as polymerases, kinases and proteases have been targets for inhibition. In contrast, of the approximately 8 distinct proteins encoded by the HPV genome, the E1 helicase is the only one with enzymatic activity (Fields et a., 1996, Fields Virology, 3rd Ed. Lippincoff-Raven, Philadelphia, Chap. 65 and refs. therein). E1 displays ori-specific DNA-binding activity, E2-binding activity, ATPase activity, and DNA helicase activity—all of which can be assayed independently for potential inhibitors. In addition, it is the most highly conserved of all papillomavirus proteins, so an inhibitor of E1 would likely be effective against multiple HPV types.
High throughput screens are known that allow the discovery of inhibitors of the helicase activity of E1 (WO 99/57283, Nov. 11, 1999). Even though ATP is needed to drive E1 helicase activity and is included in the reaction, this helicase assay cannot be used to identify competitive inhibitors of E1 ATPase function. This is a direct result of very low Km of the ATPase, for example approximately 10 μM for HPV-11 E1, and the fact that the helicase assay is routinely run with 300 μm-1 mM ATP). A more sensitive assay must be developed if the ATP binding site of E1 is to be targeted for inhibition.
Existing ATPase Assays
Helicase activity is virtually always associated with nucleoside triphosphatase activity (Matson et al., Ann. Rev. Biochem., 1990, 59, 289). Enzymatic ATP hydrolysis has been measured by a variety of methods, including colorimetric reactions; in all cases, enzymatic reactions are performed according to enzyme-specific protocols where reaction conditions are not dependent on the detection procedure (except for the inclusion of radiolabeled ATP). The detection procedure differs for the different assays in the following ways:
TLC:
The inclusion of [α-33P] or [γ-33P] ATP in the substrate for an ATPase reaction results in the release of radiolabeled phosphate or ADP. Because of their different polarity, [33P]-labeled ATP, ADP and phosphate can also be separated by thin layer chromatography (Bronnikov et al., Anal. Biochem., 1983, 131, 69) in a running solvent (e.g. lithium chloride/formic acid). The two species migrate at different distances on a TLC plate based on their relative affinities for the polar mobile phase and non-polar solid phase. Results are analyzed by scintillation counting or PhosphorImager analysis.
Although the TLC assay for quantification of released phosphate produces accurate data for ATPase activity and inhibition, it is unsuited for the mass-screening of potential inhibitors. The spotting and running of large numbers of TLC plates is time-consuming and labor-intensive. A method that lends itself to 96-well plate format and rapid quantification is needed if an ATPase assay is to be implemented in HTS format.
Charcoal:
ATP binds to charcoal but orthophosphate does not (Zimmerman et al. J. Biol. Chem. 1961, 236 (5), 1480). Thus if a reaction is run using γ-labeled ATP and charcoal is added, the starting material is adsorbed, but the product remains in solution. One can run this as a 96-well plate assay by filtering solutions through charcoal-containing filter plates, and counting the flow-through. This is not likely to be highly reproducible, and is not amenable to robotic screening.
Coupled-Enzyme Assays:
There are a number of related procedures in which another reaction is carried out on the phosphate product by a second enzyme (Rieger et al., 1997, Anal. Biochem. 246, 86 & refs. contained therein). These assays are very useful for kinetic studies, because absorbance change is generated continuously over the course of the assay, so that the reaction course can be monitored without removing aliquots as necessary for the other methods above (the distinction between continuous and stop-time assays). These methods are not significantly more sensitive than the molybdate assay (below) however, and screening results would be further complicated by the possibility of false positives being inhibitors of the coupling enzyme.
Molybdate:
Ammonium molybdate forms a complex only with phosphate to form phosphomolybdate. Pyrophosphate, nucleotide triphosphates, or other phosphate-containing molecules resulting from the reaction do not interact with molybdenum oxides. Most of the calorimetric reactions are based the formation of a complex between phosphate and the molybdate ion in acid solution, followed by reduction or binding to dyes that form colored complexes. Many variations to these techniques have been introduced with the goal of increasing sensitivity and color stability, and decreasing the amount of spontaneous ATP hydrolysis that occurs during the color-developing incubation (Gonzalez-Romo et al., Anal. Biochem. 1992, 200, 235). For instance, the phosphomolybdate complex can be reduced by ascorbic acid to generate a blue molybdenum chromogen with maximum absorbance at 700 nm (Hergenrother et al., Anal. Biochem. 1997, 251, 45). Another method is based on the formation of a brilliant green complex with malachite green in an acid medium, which has a maximum absorbance at 650 nm (Moslen et al., Anal. Biochem. 1983, 131, 69).
In fact, the malachite green assay was previously evaluated as a potential test for the ATPase activity of E1, but was found to be unsuitable because it could not accurately detect concentrations of phosphate lower than 25 μM. This presented a problem, because detection of competitive inhibitors is optimal at substrate concentrations below the Km of an enzyme. As previously mentioned, the Km (ATP) of the HPV-11 E1 ATPase has been shown to be about 10 μM, so the E1 ATPase reaction is routinely carried out at around 1-10 μM ATP. In addition, substrate consumption in an inhibition experiment is kept below 30%, so that substrate concentration remains essentially constant over the time of the reaction. The result is that 3 μM is the maximum concentration of phosphate that is released—well below the 25 μM detection limit of the malachite green assay.
All these calorimetric ATPase assays require a minimum ATP concentration of several hundred micromolar. The value of Km(ATP) for HPV-11 E1 being approximately 10 μM (measured in the absence of DNA), thus to effectively screen for competitive inhibitors of E1 ATPase activity, one should perform assays using [ATP] <10 μM.
Adsorption of Phosphomolybdate on Solid Support:
Phosphomolybdate is a large heteropolymolybdate, with a stoichiometry of [PMo12O40]3−. Because of its relatively low charge, it can be extracted from aqueous solution into organic solvents or adsorbed onto a hydrophobic surface such as Sephadex beads or nitrocellulose filters. Ohnishi et al. (J. Solid-Phase Biochem. 1976, 1(4), 287) and Ohnishi (Anal. Biochem. 1978, 86, 201) disclose a method for isolating the phosphomolybdate complex from solution by affinity chromatography on polyvinyl polypyrrolidone (PVPP) column. PVPP acts as a catalyst for the complexing reaction between PO4 and molybdenum and thereby selectively adsorbs the complex over other phosphate-containing molecules. Phosphate may be radioactively labeled and eluted from the column for counting of radioactivity. This method is limited by the fact that the labeled phosphate needs to be separated from the reaction mixture before counting. There remains a need for a robust method for phosphate determination that is amenable to a high throughput format.
Yoshimura et al. (Anal. Biochem. 1986, 58, 591) disclose a calorimetric micro-determination of molybdenum-blue by adsorbing the complex on Sephadex gel-phase. This procedure requires reduction of the complex prior to the adsorption and measures the phosphate concentration by direct absortiometry of the heteropoly acid concentrated in the gel phase. This procedure requires separation of the gel beads from the supernatant prior to measurement by colorimetry. Although this calorimetric method allows for detection of low concentrations of phosphate, it remains unsuitable for automation.
Scintillation Proximity Assay:
Hart et al. (Molec. Immunol. 1979, 16, 265) and Hart et al. (J. Nucl. Med. 1979, 20, 1062) disclose a new method for immunoassay called “scintillation proximity assay”. This technology used scintillant latex particles coated with a ligand that specifically binds an organic reactant being investigated. All further applications of this technology with hydrophobic beads has relied on providing a specific ligand coated on the beads to bind specifically to a molecule.
U.S. Pat. No. 4,568,649 discloses such beads coated with a specific ligand and specifies that the remaining active sites on the beads must be blocked prior to the assay to prevent the reactant of interest or others from binding directly to the beads rather than to the ligand. This disclosure leads away from the present invention.
Despite the wide applications of this technology since its inception, there has not been the slightest suggestion that this same technology could be used advantageously to detect radiolabeled phosphate through hydrophobic interaction with a phosphomolybdate complex. Applicant's use of the SPA concept in the detection of ATPase activity is founded on the observation that the hydrophobic phosphomolybdate complex binds to hydrophobic surfaces, particularly to the surface of polyvinyl toluene SPA beads, whereas the charged ATP molecule does not. Applicant has used that property to separate the orthophosphate from ATP or ADP and takes advantage of the scintillant-coated beads for measurement of radioactive orthophosphate. Applicant therefore provides a robust method for detecting and measuring orthophosphate. This assay is amenable to large scale and provides reproducible results for detection of Pi in the low nanomolar range. This method is also suitable for kinetic analysis not easily performed by prior art assays.